The present invention relates to Immunogenic complex in the form of ISCOM(trademark) (complexes as described for example in U.S. Pat. No. 4,578,269) and/or ISCOM(trademark) matrix (complexes as described for example in U.S. Pat. No. 5,679,354) and mucus targeting molecules for use for preparing vaccines and immune stimulating compositions for oral, nasal, urogenital and/or rectal administration.
Immunization strategies have been available for many years. However, there has been a remarkable lack of success in vaccination to control mucosal diseases. Earlier findings indicate that antigen presentation of non-replicating antigens via the mucosal surface is an inefficient means of immune response stimulation and that novel strategies are required in this route (Novel vaccination strategies for the control of mucosal infection, Alan J. Husband, Vaccine, Vol. 11, Issue 2, 107-112). Vaccination with living attenuated microorganisms has been the only possible way of protection against mucosal diseases.
It has now unexpectedly been shown that ISCOMs(trademark) containing, and ISCOM(trademark) matrix mixed with, mucosal targeting molecules or antigens give raise to high titers of antibodies when administered to mucosas. Moreover ISCOMs(trademark), containing such mucosal targeting molecules together with antigens which do not readily immunize by the mucosal mode of administration (so called passenger antigens), give raise to an immune response even in several other mucosals remote from the site of administration.
The mucosal targeting molecules have the capacity to target the lymphatic system following mucosal administration. They may be antigens which assist passenger antigens in inducing immune response and often, they will also induce immune response to themselves. In a complex mixture of antigens, some may exhibit a capacity for being target molecules for passenger molecules as well as for themselves. The strategy of using a mixture of two or more components also involves the prospect of modulating the ensuing immune response to itself as well as to passenger antigens. The modulatory effect is particularly prominent in iscom formulations with targeting molecules exemplified by IgG2a enhancement, as well as the capacity for iscoms to enhance conversion of the mucosal antibody response to IgA in mucus which is best illustrated by the enhancing effect on the IgA response to CTB. ISCOM(trademark)-matrix simply added to other antigens and mixed as a separate entity has similar properties as well as but they are often less prominent.
Lipid-containing and quillaja saponin-containing structures such as ISCOMs(trademark) (immunostimulating complexes) and ISCOM(trademark)-matrices have been reported to be effective carriers of pharmacologically and/or immunologically active substances or molecule complexes. See for example WO-A1-90/03184. In many cases, parenteral immunization of laboratory animals with structures incorporating different antigens has been demonstrated to give rise to a stronger immune response against the antigens at issue than that which is obtained after immunization with the corresponding antigen(s) in a free form.
ISCOMs(trademark) are documented to be effective carriers in enhancing the immunogenicity of small and large molecules (antigens) using parenteral administration. It has been shown that iscoms with incorporated antigens, such as protein, according to EP 0 109 942, and ISCOMs(trademark) that are carriers for small molecules, such as small antigens and oligopeptides, according to EP 0 180 564, effectively evoke immune response toward both large and small molecules.
It has also been shown that protein antigen, such as ovalbumin (OVA), in ISCOM(trademark) is able to evoke immune response even after oral immunization that includes antibody formation, T-helper cells under MHC class-2 restriction and cytotoxic T-cells (CTL) (killer cells) under MHC class 1-restriction, but that several immunizations are required (and unrealistically high doses) (Morein, B., Lovgren, K., Ronnberg, B. Clin. Immunother. 3, 461-75, 1995). On the contrary, after giving large doses (hundreds or several hundreds of xcexcg per dose of ovalbumin orally in free form, ie unincorporated in iscom), there is either no immune response or a low one. Free ovalbumin gives rise to tolerance and suppression to a subsequent parenteral immunization.
ISCOM(trademark)-matrices (and ISCOM(trademark) have well-documented, built-in adjuvant activity that evokes antibody-mediated and cell-mediated immune response. Cell-mediated immune responses under both class 1 and class 2 MHC-restrictions are evoked by ISCOMs(trademark).
In comparison to parenteral immunization, the immune response after oral and intranasal administration of ISCOMs(trademark) has been low (Mowat et al, Immunology 72, 317-322 (1991); Mowat et al., Immunology, 80, 527 (1993)). Moreover, in applying intranasal and oral immunizations, certain antigens that are incorporated in ISCOM(trademark), such as the g-protein of the rabies virus or gp 120/160 of HIV-1, have not evoked measurable immune response in the form of serum antibody response measured in ELISA. In all likelihood this is due to the fact that only a small number of the perorally or intranasally administered particles can penetrate the mucous layer, be absorbed by the intestinal epithelium barrier or M-cells of Peyers patches and there come in contact locally with the immune system.
Problems involving limited absorption and insufficient antigen presentation caused by the inability of antigens to penetrate the mucous barrier and target antigen presenting cells (APC), lymphatic tissue and/or the epithelium layer, eg Peyer""s patches (PP) or Lamina propria (LP) in the intestines, or lymphatic tissues in the tonsil region in the pharynx or other mucous-coated surfaces. Mucosal immune response will generally only occur when mucosal administration is carried out locally. ISCOMs(trademark), like other antigen-presented structures, have a limited ability to penetrate mucus, and, in varying degrees, a limited ability to bind and be absorbed by the epithelium, or to reach and be absorbed by M-cells in PP to thereafter be forwarded to antigen-presented cells (APC) and to stimulate lymphocyte populations. Even when administration is done into the nasal mucosa, the targeting and antigen uptake by APC and the induction of the immune response is insufficient when using current technology. CT and LT have been used as mucus targeting molecules but due to high toxicity those are not suitable for use in man and animals. The B-subunit of these toxins are not toxic but very much less effective and for that reason not practically suitable for prophylactic or clinical use. There is thus a need for better presentation systems for antigens that are intended for use in oral or nasal administration. An important reason why there is this need for better antigen-presenting systems that are effective in administration via mucosal membranes is that this kind of administration evokes a local immune response in the membranes. Although it is known that common lymphatic systems exist where immunization e.g. in the gut will result in immune responses in remote mucosal surfaces via gut associated lymphatic tissue (GALT) or immunization in the respiratory tract with result in immune response in other mucosal surfaces via broncho-associated lymphatic tissue (BALT) these responses are generally low and mucus targeting molecules or antigen presenting systems promoting remote mucosal targeting is not particularly defined. A number of infections occur via mucosal membranes in places like the respiratory passages, the intestinal tracts or the genital tract, and a first immune defence barrier exists in the mucosal membranes. Moreover, both oral and nasal administrations in contrast to parenteral administration by injection have the advantage of not requiring medically-trained personnel.
The invention relates to Immunogenic complex in the form of ISCOM(trademark) and/or ISCOM(trademark) matrix and mucus targeting molecules for use for preparing vaccines and immune stimulating compositions for oral, nasal, urogenital and/or rectal administration.
It has been shown that by using immunoenhancing complexes chose from ISCOMs(trademark) and ISCOM(trademark) matrices contained at least one glycoside, at least one lipid of which one being cholesterol
a) at least one mucus targeting molecule chosen from substances that target lymphatic tissue and thereby induce immune response in local mucosal membranes; and possibly
b) at least one passenger antigen chosen from pharmacological immuno-affecting or enhancing or immunogenic substances that do not easily reach lymphatic tissue through mucous membranes, vaccines and immunostimulating agents can be prepared for oral, nasal, urogenital and/or rectal administration. This will provide antibodies against the passenger antigen in serum, local IgA immune response in secretions in the local membrane at the site of the administration and also in membranes in other places, particularly in the respiratory tract in the intestines and in the genital tract.
One aspect of the invention relates to the use of ISCOM(trademark) complexes containing at least one mucus targeting molecules for preparing a vaccine or immune modulating composition directed against that molecule. The ISCOM(trademark) may also contain at least one passenger antigen together with the mucus targeting molecule or molecules in order to give raise to an immune response against the targeting molecule(s) and the passenger antigen(s).
The invention also relates to the use of ISCOM(trademark) matrices that are mixed with at least one mucus targeting molecule for preparing a vaccine or immune modulating composition directed against that molecule. The ISCOM(trademark) matrices may also be mixed with at least one passenger antigen together with the mucus targeting molecule or molecules in order to give raise to an immune response against the targeting molecule(s) and the passenger antigen(s).
ISCOM(trademark) contains at least on glycoside, at least one lipid and at least one type of antigenic substances, especially proteins and peptides. These complexes enhance the immunogenicity of the administered antigens and may contain one or more immunomodulating (adjuvant-active) substances as described in EP 0 109 942 B1, EP 0 242 380 B1 and EP 0 180 564 B1.
Because certain antigens do not require physical integration in the ISCOM(trademark) particle, great advantages are also to be gained from local mucosal administration using ISCOM(trademark)-matrix mixed with passenger antigen in separate entities. Such antigen is exemplified by gB2 of Herpes simplex 2 virus, which induce immune response by mucosal administration but the immune response is considerably enhanced by both matrix and iscom.
Matrices contain at least on glycoside that is an adjuvant-active substance and at least one lipid. Matrices have an immunostimulating effect on administration together with antigenic substances, see EP 0 436 620 B1. The matrices may have various kinds of immunoactive, immunoenhancing components (see EP).
According to the invention, complexes may be composed of an ISCOM(trademark) complex with a mucus targeting molecule and/or passenger molecule(s) (that are) integrated into the ISCOM(trademark) complex or else tied to a ready-made iscom complex.
They may also be made up of ISCOM(trademark) matrices to which the mucus targeting and passenger antigens can be chemically coupled or bound by hydrophobic interactions, making them ISCOM(trademark) or ISCOM(trademark) matrices that are separated entities mixed with the antigen formula.
According to the invention, these complexes can be used to prepare vaccines and immunostimulating agents for oral, nasal or rectal modes of administration, or other types of local mucosal administration. They induce immune response in other mucous membranes than the ones they are administrated in, such as the urogenitals, the intestines, the upper respiratory tracts and the lungs as well as circulating antibodies.
According to the invention, these complexes also modulate the immune response to encompass strong specific IgA in various mucosal secretions and to encompass an increased IgG2a response in serum indicating a participation of interferon-gamma and T-helper 1 response.
ISCOM(trademark), like ISCOM(trademark) matrices, can, according to the invention, modulate antibody-mediated immune responses and the cell-mediated immune response that differ from immunomodulation evoked by a parenteral mode of immunization. Local mucosal administration also gives rise to immune response against other antigens or antigen determinants. From this point of view, new possibilities arise to elicit immune response that is not recognizable in parenteral immunization, eg against carbohydrate structures.
It is also of interest to use them in the preparation of vaccines intended for immunotherapy or for the treatment of allergies e.g. by desensitation via immunotherapy. They are also useful in breaking tolerance to antigens induced by CTB or LTB or other tolerance conditions caused by local application.
Mucus targeting molecules and passenger antigens are derived from microorganisms such as bacteria, viruses or parasites, in particular those named in EP 0 109 942 B1.
Mucus targeting molecules are substances that target lymphatic tissue to induce immune response in local mucosal administration in various mucosal surfaces as well as systemic immune response. In particular, antigen substances such as proteins, peptides, carbohydrates are intended. The carbohydrates may be polysacharides, glucolipides or glucopeptides.
Mucus targeting molecules may be of bacterial or viral origin, such as the bacterial cholera toxin and its subunit B (CTB), or the heat-labile toxin i E-coli and its subunit B (LTB). Other examples are enveloped proteins from viruses or proteins from bacteria, which can penetrate the mucosal membranes and infect the respiratory passages, such as the influenza virus, respiratory syncytial virus (RSV), corona virus, herpes viruses, pox virus, membrane proteins from Mycoplasma and fimbriae from bacteria such as hexons and pentons from adenovirus, Norwalk virus, rotavirus, and enterovirus from the family of picornaviridae and astrovirus. Other examples of usable antigens are the enveloped proteins from viruses and bacteria that infect the intestines, such as influenza virus adenovirus, reoviruses, corona virus and fimbriae from Excherichia coli (K88, K99, K981-B), Shigella, Clamydia and membrane proteins from Mycoplasma.
Such microorganisms can be the measles, German measles and chicken pox viruses; Mycoplasma pneumoniae, Mycoplasma mycoides, Chlamydia pneumoniae, Neisseria meningitidis, Neisseria gonorrhoea, Vibrio cholerae, Salmonella typhi, Streptococcus mutans, Helicobacter pylori, Streptococcus pyogenes, Corynebacterium diphtheriae, Mycobecterium tuberculosis, Yersinia pestis, Salmonella typhi, Borrelia burgdorferi, Plasmodium vivax, Plasmodium falciparium, Toxoplasma gondii, Trypanosoma cruzi Trypanosoma brucei, Gardia lambiia and Entamoeba histolytica; and Cryptococcus neoformans and Histoplasma capsulatum, Pneumococcus pneumonia, Haemophilus influenzae. 
Passenger antigens may be chosen either from the group of pharmacological antigen substances or from antigen substances that are relevant from immunization and that lack tropism for mucous membranes, i.e., less effectively penetrate mucous membranes, for instance gB and gD in various Herpes viruses including Herpes Simplex 1 and 2, bovine herpes virus 1, picorna virus, gp 120 and gp 160 in HIV-1 or the corresponding envelope protein in HIV-2, as well as from other retroviruses, hepadna viruses e.g. pox virus, adenoviruses, cardivirus, togavirus, flavavirus, irdiovirus, birnavirus, provovirus, popovavirus, picornavirus, calicivirus, astrovirus, arenavirus, bunyavirus, ortomyxovirus, paramyxovirus, G-protein from the rabies virus, or from bacteria such as salmonella, E. coli, Shigella, Chlamydia, ricketsia, bartonella or Mycoplasma. They may also be various recombinant products or synthetic antigens that lose or do not lose their capacity after cloning or that increase their capacity to induce immune response via incorporation in iscom with mucus targeting molecules or with matrices since they are mucus targeting to the lymphatic tissue when applied on mucous membranes.
In those cases where the complexes are ISCOMs(trademark), they may be prepared as described in the European patent EP 0 109 942 B1. Here, viruses, mycoplasma, bacteria, parasites and animal cells are mixed, containing antigens or antigenic determinants, particularly proteins or peptides or isolated molecules that have hydrophobic or amphiphilic regions, are mixed with one or more solubilizing agents, after which the antigens or determinants are separated from the solubilizing agent in the present of, or is separated from the solubilizing agent and is directly transferred to a glycoside solution, containing cholesterol, phospholipids and one or more glycosides with hydrophobic or hydrophilic domains in a concentration of at least a minimum of the critical micellar concentration, after which a protein complex is formed and then isolated and purified.
One can start from whole microorganisms containing passenger, antigen and/or mucus targeting molecules, whereby mucus targeting molecules or passenger antigens that are either more or less purely prepared, synthetic or prepared using a hybrid-DNA technique may be added. The additive may be put in at any partial stage, as is explained in EP 0 109 942 B1, especially on pp. 4-8. The preferred procedure is to put in the additive before adding the lipids and glycosides.
Furthermore, you can use a base consisting of mucus targeting molecules or passenger antigens that are more or less purified, synthetisized chemically or prepared using a hybrid-DNA technique according to EP 0 109 942 B1 as on pp. 4-8. In this case, it is appropriate to add the lipids before the complex has been isolated and cleaned, as is described in the applicant""s European patent EP 0 242 380 B1.
The lipids used are particularly the kind described in the applicant""s patent EP 0 109 942 B1 in particular on p. 3 and in EP 0 436 620 B1, p. 7, lines 7-24. Sterols such as cholesterol and phospholipids such as phosphatidylethanolamine and phosphatidylcholine GM1, where particularly used.
The lipids may also include lipid-containing substances that bind to cell-forming components, for instance glycolipids such as the receptor of cholera toxin, the gangliosid GM1, and focused blood group antigen. The cell-binding components can then function as mucus targeting molecules and be tied to the lipid-containing substances by simply mixing with complexes that contain them.
It is also possible to first prepare ISCOM(trademark) particles from a mucus targeting or passenger antigen and then add on the passenger and mucus targeting antigens respectively using current conjugation methods, preferably chemical coupling methods, as is described in the applicant""s European patents EP 0 180 564 B1 and EP 0 436 620 B1.
The base could also be matrices that are prepared by solubilizing at least one sterol in a solubilizing agent, adding the glycoside or the saponins and the other lipids, after which the solution agent may be removed if desired, i.e. if it cannot be accepted in the final product. Matrices are usually transferred to a water solution in which the individual parts are not dissolvable. The solubilizing agent can be removed, for example, by gel filtration, ultrafiltration, dialysis or electrofores. The matrices may then be purified from an excess of sterol and saponin by means of, for example, centrifugation through a density gradient or by gel centrifugation.
The solubilizing agent may be any of those mentioned in EP 0 436 629 B1, p. 5, lines 24-45. The other components and the preparation process are also described in this document.
Passenger and mucus targeting molecule can be bound to the matrices using current binding-conjugation methods, see above. It is also feasible to mix adjuvant molecules and/or passenger antigens with an ISCOM(trademark) molecule in which the passenger antigen or the adjuvant molecule has been integrated, or with ISCOM(trademark) and/or matrix complexes to which the passenger antigen or the mucus targeting molecule has been connected. It is also possible to mix both mucus targeting molecule and passenger antigen with an ISCOM(trademark) complex or matrix in a separate entity, in which case the ISCOM(trademark) complex contains a different antigen molecule.
The glycosides used in the preparation may be those described in EP 0 109 942 B1, p. 4, last paragraph. The preferred method is to use saponins such as triterpensaponins, particularly Quil A or defined components of it, particularly those described in the applicant""s European patent EP 0 436 620 B1 p. 4, lines 19-46. These may by QHA, QHB, QHC or other compositions of Quil A, such as 703. Glycosides are adjuvants. Or such components described by Kensil, Kersten or Dalsgaard (Kensil, C. R., Patel, U., Lennick, M. and Marciani, D., J. Immunol, 146, 431-437, 1991; Kersten, G. G. A., Spiekstra, A., Beuvery, E. C. and Commelin, D. J. A. BBA1062, 165-171, 1991; or patent WO95/09179 Dalsgaard). It is also possible to incorporate other adjuvants or immune-modulating components than the glycosides in the ISCOMs(trademark) or in the matrices as mentioned in EP 0 436 620 B1.
Examples of such adjuvants are provided in Cox et al., CRS, 1992. The preferred method is to use MDP, MTP and avridin.
If the mucus targeting molecule(s) or the passenger antigen(s) are lacking hydrophobic or amphiphatic groups, they can be added on so that the antigen can bind to the ISCOM(trademark) particle. Examples of such groups are to be found in EP 0 242 380 B1 p. 9 and in EP 0 436 620 B1 p. 6, line 33 to p. 7, line 6, where the connecting methods are described. They may be lipids as in Example 2 below.
The relative amounts of cholesterol, lipids and antigen that can be used are seen in the above-mentioned patents EP 0 109 942 B1, EP 0 180 564 B1, EP 0 242 380 B1 and EP 0 436 620 B1.
Of the bacterial mucus targeting molecules, it is the cholera toxin, its subunits such as B (CTB) and the heat-labile toxin in E.coli with its subunits such as B (LTB) that are the most preferable.
Cholera is one of the most dangerous of all diarrhea diseases and is caused by the Vibrio cholera-bacteria 0 group 1. These bacteria colonize in humans in the small intestine where they secrete an exotoxin protein known as the cholera toxin.
Similar diseases are caused by so-called xe2x80x9centerotoxicxe2x80x9d coli bacteria (ET) although these symptoms are usually milder and are caused by a heat-labile toxin (LT) that is similar to the cholera toxin (CT). These toxins are so similar that the y bind to the same receptor.
The structures of CT and LT are well defined regarding structure and function. They are oligomer proteins containing a part that binds to the cholera toxin receptor, namely the B-subunit, which in turn contains five subentities that each have an approximate mole weight of 11,600 and have the form of pentamerous rings. The A subunits are proteolytic split polypeptides with a mole weight of approximately 28,000 consisting of two disulfide-bound fragments. The larger A1-fragment contains the toxin-enzyme activity while the smaller A2-fragment attaches the A1-fragment to the B5 ring. CT binds with close affinity to a class of receptors that exist on the surface of the so-called brush-border membrane in the small intestine, and to the plasma membrane in most mammalian cells as well. The receptors are composed of the gagliosid GM1. Moreover, LT binds as well to an unnamed glycoprotein (Holmgran et al., Infect. Immun., 38, 424-433 (1982)). These proteins can also be incorporated in iscom or bound to iscom or matrices, serving as binding molecules to LT.
The gangliosid GM1 and other receptors for mucus targeting molecules of lipid type or hydrophobic may be included in the iscom particles lipid containing region. They may even be a part of the lipid composition in the matrices.
When the receptor is incorporated in the iscom particle or the iscom matrix particle, it can be mixed with a corresponding ligand which binds to the receptor incorporated in the particle. In cases where the carrier or passenger antigen""s receptor is a gangliosid, the antigen can be bound to such a receptor through a simple mixing procedure. This procedure is explained in Example 1 below. Similarly, other receptors will bind its antagonist.
The cholera toxin contains the A subunit, which exerts toxin activity, and the B subentity which attach the toxin to the plasma membrane on the cell via a glycolipid (GM1). To reduce the toxicity, usually only the B subunit in the cholera toxin (CTB) is used as a vaccine antigen for producing immune response. The B subunit is not toxic and evokes a relatively weak immune response as compared to CT in local intranasal and parenteral immunization, for instance subcutaneous or intramuscular immunization. In other words, the B subentity has low adjuvant activity. CTB is also available as a recombinant DNA-product. (rCTB) (EP 368 819).
It is difficult to obtain worthwhile physical or economic yields by binding antigens covalently to CTB and LTB, because only a limited number of amino and/or carboxy groups can be activated without seriously diminishing CTB""s and LTB""s antigen activity or their ability to mucus targeting in the mucosal membranes, and their ability to localize themselves and the enclosed antigens to the lymphatic organs and cells to evoke immune response. Even if a sufficient number of groups are available for chemical conjugating on a carrier molecule, it is well known that it is difficult to obtain worthwhile physical and economic yield from such constructions (Lxc3x6vgren et al., Immunol. Methods 173, 237-243).
The use of mucus targeting molecules in ISCOMs(trademark) has many advantages. This holds especially true in the preparation of oral, nasal or rectal vaccines against various diseases. Such vaccines can contain a carrier construction with antigen, adjuvant component(s) supplemented with, for example, CTB or LTB in order to localize the construction to the lymphatic organs and cells in the intestinal canal and to other mucous membranes via GALT MALT, NALT (Gut, Mucosal and Nasal-pharyngeal Associated Lymphatic Tissue), such as in the respiratory tracts or directly through local administration.
The ISCOM(trademark) being larger than the mucus targeting molecules there is room for both incorporating chosen passenger antigens and chosen adjuvant components, conjugation or in some other way, such as through hydrophobic interactions or electrostatic binding.
In producing matrices, a common ratio of sterol, another lipid, and glycoside is 0.2-10:0.2-10:1-100, preferably 1:1:5. If using a lipid-containing receptor, it can replace the other lipid completely so that the ratio of sterol, lipid-containing receptor and glycoside will be as above. Another possibility is to use both the lipid-containing receptor and another lipid, preferably phosphatidylcholine or phosphatidylethanolamine plus the receptor, so that the ratio will be sterol; other lipid:receptor:glycoside 0.2-10:0.2-10:0.1-1:5-10, preferably 1:1:0.25:5. The number of receptor molecules depend on the number of molecules one wishes to add on.
In principle, the components can be put in at any ratio whatsoever. It has been shown that the finished product receives the weight ratio between the various components as given above, and that the excess does not enter in. If too much of the other lipid(s) are used, the complex becomes xe2x80x9cfattyxe2x80x9d and fragile and crumbles easily. Too little of the other lipid leads to the complexes not being formed and annular (ring-shaped) subunits being formed instead. This can be determined through electron microscopy.
It is possible to determine whether the ISCOM(trademark) or matrix has been obtained by examining the product in an electron microscope. Typical matrices or ISCOMs(trademark) have a characterically open, spheric structure consisting of circular subunits, as can be seen in FIG. 3 in EP 0 109 942 B1 and FIG. 3 in this application. The ISCOMs(trademark) have a lower sedimentation constant than corresponding micelles and often a higher sedimentation constant than the corresponding monomeric forms of protein or peptide. Matrices and ISCOMs(trademark) have a sedimentation constant of approximately 12-22 S, in particular 20 S.
The advantage of using lipid-containing receptors for binding the target or mucus targeting antigens is that the matrices can be prepared with glycoside, sterol, possibly another lipid and a lipid-containing receptor, and then simply mixed with the mucus targeting molecule. The process is more effective, less expensive and simpler than if one were to prepare ready-made iscom containing the ingredients above plus the mucus targeting protein antigen or if chemical coupling methods were used to connect the antigen to the ready-made matrix.
When antigen is integrated into ISCOM(trademark) or combined with matrix using chemical coupling methods, the amino or the carboxyl groups that make up the antigen determinants are modified.
Antigen determinants are denatured when the antigen is activated to covalently bind it to the iscom or to attach a hydrophobic tail to facilitate for integration into iscom or when it is coupled by chemical methods to matrix or iscom (when two antigens are used.) This means that the amount of unmodified antigen is greatly reduced compared with a mode of allowing the antigen to bind to a lipid-containing receptor. This can mean, for example, that approximately five times more antigen is needed compared to the mode of using a lipid-containing receptor. When a lipid-containing receptor is used, the process is much less expensive. Parallel to a decrease in the degree of incorporation, there is an increase in the amount of glycosid and the adjuvant content, which partly compensates for the smaller amount of antigen in the attained immune response. At the same time, toxicity may increase due to the increased adjuvant content. In principle, however, the immune response is higher when receptor binding of the antigen is used.
There is another advantage, too, when aspiring to bind a mucus targeting molecule and a passenger antigen to ISCOM(trademark) or matrix. If the ISCOM(trademark) or matrix is made from lipid-containing receptors, there is more space to integrate the passenger antigen into iscom or to bind it to matrix using chemical coupling methods. The usage of a lipid-containing receptor does not influence the binding of the passenger antigen. This makes it easier to obtain optimal ratios. It is easier to regulate the amount of passenger antigen and mucus targeting molecules integrated into ISCOM(trademark) or connected to ISCOM(trademark) or matrix using chemical methods. On the other hand, if iscom is made using a mucus targeting molecule and antigen with the same methods, they compete for the binding and it becomes difficult to wholly regulate the incorporation of both components.
Particularly in regard to the cholera CTB subunit, which has five binding sites subunits, it is possible to bind up to 13 times the weight of the GM1-receptor. This still leaves binding entities in CTB that are able to bind to receptors in the mucous membrane and serve as molecules.
The weight ratio of sterol, other lipid, protein and glycoside is 0.2-10:0.2-10:2-10:1-100, preferably 1:1:1:5-10 in subcutaneous administration. In oral or intranasal administration, the amount of glycoside may be higher in the ratio above, namely 1-200, preferably 5-20.
These are the appropriate amounts both when matrix is first produced and bound to the antigens using chemical coupling methods and later when iscom particles are made.
All of the above-mentioned paten documents as well as the priority document SE 9600647-3 are included as reference.
The amount of ISCOM(trademark), matrix and antigen is chosen to be pharmaceutically effective and can be estimated by the man of art. For humans at least 1 xcexcg, especially from 1 up to 200 xcexcg of the antigen may be used, whereby economical aspects set the upper limit. For animals the dose may be at least 0.1 xcexcg of the antigen depending on the antigen and the size of the individual.
ISCOM(trademark) or ISCOM(trademark) matrix can be prepared in compositions containing a solubilizing agent, e.g. water or sodium chloride. The composition may also contain the detergent used in making the complex as a solubilizing agent, if it is acceptable from the point of view of human or veterinary medical practice. Moreover, the compositions may contain other additives and filler agents acceptable to human or veterinary medical practice.
Such a composition may contain, for example, an ISCOM(trademark) complex and an inert filler such as sodium chloride. It may also consist of a matrix mixed with antigen.
The vaccine may be made available for modes of administration that contain an entity with matrix in a composition containing an inert filler and an entity with the antigen in a composition containing an inert filler. These two compositions are intended to be administered at the same time.
EXP OVA I